Published OnlineFirst October 26, 2016; DOI: 10.1158/2159-8290.CD-15-1263 RESEARCH ARTICLE The Master Neural Transcription Factor BRN2 Is an Androgen Receptor–Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer Jennifer L. Bishop1, Daksh Thaper1,2, Sepideh Vahid1,2, Alastair Davies1, Kirsi Ketola1, Hidetoshi Kuruma1, Randy Jama1, Ka Mun Nip1,2, Arkhjamil Angeles1, Fraser Johnson1, Alexander W. Wyatt1,2, Ladan Fazli1,2, Martin E. Gleave1,2, Dong Lin1, Mark A. Rubin3, Colin C. Collins1,2, Yuzhuo Wang1,2, Himisha Beltran3, and Amina Zoubeidi1,2 ABSTRACT Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor–resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo. Mecha- nistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with clas- sic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors. SIGNIFICANCE: Understanding the contribution of the AR to the emergence of highly lethal, drug- resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discovery; 7(1); 1–18. ©2016 AACR. 1Vancouver Prostate Centre, Vancouver, British Columbia, Canada. Corresponding Author: Amina Zoubeidi, Vancouver Prostate Centre, 2660 2Department of Urologic Sciences, Faculty of Medicine, University of Oak Street, Vancouver, BC V6H3Z6, Canada. Phone: 604-809-4947; Fax: British Columbia, Vancouver, British Columbia, Canada. 3Department of 604-875-5654; E-mail: [email protected] Medicine, Division of Hematology and Medical Oncology, Weill Cornell doi: 10.1158/2159-8290.CD-15-1263 Medical College, New York, New York. ©2016 American Association for Cancer Research. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). OF1 | CANCER DISCOVERY JANUARY 2017 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 26, 2016; DOI: 10.1158/2159-8290.CD-15-1263 INTRODUCTION for neuroendocrine (NE) markers that characterize NEPC, it is often distinguished from prostatic adenocarcinoma by Progression from primary prostate cancer to advanced reduced AR expression or activity (5). Clinical presentation metastatic disease is heavily dependent on the androgen of NEPC reflects this shift away from reliance on the AR, as receptor (AR), which fuels tumor survival. In men whose patients typically present with low circulating levels of PSA treatments for localized prostate tumors have failed, or despite high metastatic burden in soft tissues, and are refrac- in those who present with metastatic disease, androgen tory to APIs (3). Importantly, it has been reported that under deprivation therapies (ADT) are used to deplete circulating the strong selective pressure of potent APIs like ENZ, these androgens to abrogate AR signaling and prevent disease “non–AR-driven” prostate cancers, which include NEPC, progression. Eventually, however, prostate cancer recurs may constitute up to 25% of advanced, drug-resistant CRPC after first-line ADT as castration-resistant prostate cancer cases (6). Not surprisingly, therefore, the incidence of NEPC (CRPC). Despite low levels of serum androgens in men with has significantly increased in recent years (7), coinciding with CRPC, reactivation of the AR occurs; thus, it remains central the widespread clinical use of APIs. to tumor cell survival, proliferation, and metastatic spread. A number of molecular mechanisms likely facilitate the Targeting the AR is a cornerstone therapeutic intervention progression of CRPC to NEPC. These include loss of tumor in patients with CRPC, and AR pathway inhibitors (API) suppressors, such as RB1 (8, 9) and p53 (10), amplification that further prevent AR activation, such as enzalutamide of MYCN (11), mitotic deregulation through AURKA (11) (ENZ), have become mainstays in the prostate cancer treat- and PEG10 (12), epigenetic controls such as REST (13–15) ment landscape (1). Despite its being a potent API, the treat- and EZH2 (11, 16), and splicing factors like SSRM4 (14, 17). ment benefits of ENZ are short-lived in patients with CRPC Importantly, the AR plays a crucial, albeit still mechanisti- and resistance rapidly occurs (2). cally unclear, role in NEPC. Reports over many years have ENZ-resistant (ENZR) CRPC represents a significant clini- highlighted how ADT (18, 19) or loss of AR promotes the NE cal challenge not only due to the lack of third-line treatment differentiation of prostate cancer cells (reviewed in ref. 20); as options to prevent AR-driven tumor progression but also such, many genes associated with an NE phenotype, includ- because it can be a precursor to rapidly progressing and ing ARG2 (21), HASH1 (22), and REST (14, 15) are controlled lethal neuroendocrine prostate cancer (NEPC). Although by the AR. Although this evidence underscores an inverse NEPC can rarely arise de novo, it is increasingly defined as correlation between AR expression and/or activity and mol- a variant of highly API-resistant CRPC (3, 4). Aside from ecular events leading to NEPC, the mechanisms by which the the unique small-cell morphology and positive staining AR directly influences the induction of an NEPC phenotype JANUARY 2017 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 26, 2016; DOI: 10.1158/2159-8290.CD-15-1263 RESEARCH ARTICLE Bishop et al. from CRPC under the selective pressure of APIs such as ENZ 42DENZR, 42FENZR, 49CENZR, 49FENZR, etc.; Supplementary remain elusive. Fig. S1A–S1C) and were screened for protein expression of Answering such questions requires a model of API-resist- AR and PSA. Reflectingin vivo data, the established cell lines ant CRPC that recapitulates the transdifferentiation of ade- displayed heterogeneous expression of PSA, yet all retained nocarcinoma to NEPC that occurs in patients. Herein, we expression of the AR (Fig. 1G). Importantly, 42DENZR present an in vivo–derived model of ENZR, which, different and 42FENZR cell lines derived from PSA- tumors (Fig. 1E from others (23–25), underscores the emergence of tumors and Supplementary Fig. S1C and S1D) remained PSA- in vitro with heterogeneous mechanisms of resistance to ENZ over (Fig. 1G), whereas 49CENZR and 49FENZR cells derived from multiple transplanted generations. These include the natu- PSA+ tumors (Fig. 1F; Supplementary Fig. S1B and S1D) ral acquisition of known AR mutations found in ENZR retained PSA expression (Fig. 1G). Accordingly, sequenc- patients (25–28) and the transdifferentiation of NEPC-like ing the ligand binding domain (LBD) of the AR revealed tumors through an AR+ state, without the manipulation of the presence of the F878L AR activating mutation in PSA+ oncogenes typically used to establish NEPC in murine pros- 49CENZR and 49FENZR cells but not in PSA− 42DENZR and tate cancer models (29–31). Using this model and data from 42FENZR cells (Fig. 1H). Emergence of this mutation in only patients with prostate cancer, we show that a master regula- PSA+ ENZR cells supports previous results showing this tor of neuronal differentiation, the POU-domain transcrip- alteration mediates resistance to ENZ (25–28) and suggests tion factor BRN2 (encoded by POU3F2; ref. 32), is directly that it may be one mechanism by which the AR is reactivated transcriptionally repressed by the AR, is required for the specifically in this subset of ENZR cells. expression of terminal NE markers and aggressive growth Mounting evidence suggests that “non–AR-driven” phe- of ENZR CRPC, and is highly expressed in human NEPC notypes of prostate cancer can emerge under the selective and metastatic CRPC with low circulating PSA. Beyond pressure of potent APIs like ENZ. Importantly, “non–AR- suppressing BRN2 expression and activity, we also show driven” tumors are not necessarily negative for AR expression; that the AR inhibits BRN2 regulation of SOX2, another in fact, many retain AR but show reduced AR activity (33). transcription factor associated with NEPC. These results Accordingly, our model of ENZR showed that approximately suggest that relief of AR-mediated suppression of BRN2 is a 25% of serially transplanted tumors, which emerged under consequence of ENZ treatment in CRPC that may facilitate constant presence of ENZ, may be non–AR-driven (Fig. 1D), the progression of NEPC, especially